3. RESULTS
3.1. Identification of FtsH substrate proteins by proteomics
3.1.1. Identification of the Spo0M protein as a putative substrate
To identify putative substrates of FtsH, the proteomes of a wild type ftsH and its isogenic ftsH::erm knockout strain taken at stage 0 were analyzed by 2D-gel electrophoresis and individual protein spots were identified by mass spectrometry. The protein quantity of each spot in both strains was compared and analyzed by the Delta 2D software. Approximately 50 proteins were strongly increased or decreased in the ftsH knockout strain as compared to the wild type strain (Table 3.1 and Table 3.2). According to the SubtiList functional categories, the identified proteins were classified into functional groups and most of them perform basic metabolic functions in the cell such as translation, amino acid metabolism, glycolysis and being part of the tricarboxylic acid (TCA) cycle (Table 3.3 and Table 3.4).
Results
Table 3.1. List of proteins increasing in an ftsH knockout strain No. Ratio Accession
number Protein name
1 4.54962 Spo0M Sporulation-control gene
2 3.73728 YjoA Unknown similar to unknown proteins 3 3.13626 Tkt Transketolase
4 3.04021 TufA Elongation factor Tu 5 2.84877 GlnA Glutamine synthetase
6 2.59722 YoxD Unknown similar to 3-oxoacyl- acyl-carrier protein reductase
7 2.38021 SodA Superoxide dismutase
8 2.36637 YjbG Unknown similar to oligoendopeptidase 9 2.3513 Icd Isocitrate dehydrogenase
10 2.34449 Dat Probable D-alanine aminotransferase 11 2.31573 FabI Enoyl-acyl carrier protein reductase
12 2.31573 YvqH Unknown similar to unknown proteins from B.
subtilis
13 2.31118 Ddl D-Alanyl-D-alanine ligase A 14 2.31118 Pgk Phosphoglycerate kinase
15 2.28535 YvgN Unknown similar to dehydrogenase 16 2.27029 BkdB Lipoamide acyltransferase
17 2.19324 YceC Unknown similar to tellurium resistance protein 18 2.19095 Hag Flagellin protein
19 2.15821 Pyk Pyruvate kinase
20 2.12796 Tpx Probable thiol peroxidase
21 2.0957 Eno Enolase
22 2.0957 SucC Succinyl-CoA synthetase (beta subunit) 23 2.08144 YvaB Unknown similar to NAD(P)H dehydrogenase
(quinine)
24 2.05179 GcvPB Probable glycine decarboxylase (subunit 2) 25 2.05179 YumC Unknown similar to thioredoxin reductase 26 2.00393 AsnS Asparaginyl-tRNA synthetase
27 2.00393 CysS Cysteinyl-tRNA synthetase 28 2.00393 DhaS Aldehyde dehydrogenase
Results
Table 3.2. List of proteins decreasing in an ftsH knockout strain No. Ratio Accession
number Protein name 1 0.49203 Drm Phosphopentomutase 2 0.49203 RocD Ornithine aminotransferase
3 0.48638 GapB Glyceraldehyde-3-phosphate dehydrogenase 4 0.48287 PyrAB Carbamoyl-phosphate synthetase (catalytic
subunit)
5 0.48002 CysC Putative adenylylsulfate kinase 6 0.48002 PyrH Uridylate kinase
7 0.47701 NusG Transcription antitermination factor 8 0.46497 PnpA Polynucleotide phosphorylase (PNPase) 9 0.46428 Fmt Methionyl-tRNA formyltransferase
10 0.44283 ResD Two-component response regulator involved in aerobic and anaerobic respiration
11 0.44283 SucD Succinyl-CoA synthetase (alpha subunit) 12 0.44091 PdhC Pyruvate dehydrogenase (dihydrolipoamide
acetyltransferase E2 subunit) 13 0.43359 Upp Uracil phosphoribosyltransferase 14 0.42343 OppD
Oligopeptide ABC transporter (ATP-binding protein) (initiation of sporulation, competence developme
15 0.41514 RocG Glutamate dehydrogenase (major)
16 0.39438 OdhA 2-Oxoglutarate dehydrogenase (E1 subunit) 17 0.38879 IolD Myo-inositol catabolism
Results
Table 3.3. Functional groups of proteins increasing in the absence of FtsH according to the SubtiList database
COGs Protein
Name Functional groups
Information storage and processing COG0050J TufA Elongation factor Tu
COG0017J AsnS Asparaginyl-tRNA synthetase COG0215J CysS Cysteinyl-tRNA synthetase
Cellular processes and signaling
COG2310T YceC Unknown similar to tellurium resistance protein COG1181M Ddl D-alanyl-D-alanine ligase A
COG1344N Hag Flagellin protein
COG2077O Tpx Probable thiol peroxidase
COG0492O YumC Unknown similar to thioredoxin reductase Metabolism
COG0538C Icd Isocitrate dehydrogenase COG0508C BkdB Lipoamide acyltransferase
COG0045C SucC Succinyl-CoA synthetase (beta subunit) COG1012C DhaS Aldehyde dehydrogenase
COG0021G Tkt Transketolase
COG0126G Pgk Phosphoglycerate kinase COG0469G Pyk Pyruvate kinase
COG0148G Eno Enolase
COG0174E GlnA Glutamine synthetase
COG1003E GcvPB Probable glycine decarboxylase (subunit 2) COG0115EH Dat Probable D-alanine aminotransferase COG0623I FabI Enoyl-acyl carrier protein reductase
COG1182I YvaB Unknown similar to NAD(P)H dehydrogenase (quinone) COG0605P SodA Superoxide dismutase
General function prediction only - function unknown COG4326R Spo0M Sporulation-control gene
COG0300R YoxD Unknown similar to 3-oxoacyl- acyl-carrier protein reductase
- NA - YjbG Unknown similar to oligoendopeptidase
- NA - YvqH Unknown similar to unknown proteins from B. subtilis COG0656R YvgN Unknown similar to dehydrogenase
COG2318S YjoA Unknown similar to unknown proteins COG: Clusters of Orthologous Groups; NA: Not Available
Results
Table 3.4. Functional groups of proteins decreasing in the absence of FtsH according to SubtiList database
COGs Protein
Name Functional groups
Information storage and processing COG1185J PnpA Polynucleotide phosphorylase (PNPase) COG0223J Fmt Methionyl-tRNA formyltransferase COG0250K NusG Transcription antitermination factor Cellular processes and signaling
COG0745TK ResD Two-component response regulator involved in aerobic and anaerobic respiration
Metabolism
COG0074C SucD Succinyl-CoA synthetase (alpha subunit) COG0508C PdhC Pyruvate dehydrogenase (dihydrolipoamide
acetyltransferase E2 subunit)
COG0567C OdhA 2-Oxoglutarate dehydrogenase (E1 subunit) COG1015G Drm Phosphopentomutase
COG0057G GapB Glyceraldehyde-3-phosphate dehydrogenase COG4992E RocD Ornithine aminotransferase
COG0444EP OppD Oligopeptide ABC transporter (ATP-binding protein) (initiation of sporulation, competence developme COG0334E RocG Glutamate dehydrogenase (major)
COG3962E IolD Myo-inositol catabolism
COG0458EF PyrAB Carbamoyl-phosphate synthetase (catalytic subunit) COG0528F PyrH Uridylate kinase
COG0035F Upp Uracil phosphoribosyltransferase COG0529P CysC Probable adenylylsulfate kinase
The proteomic approach was used to identify FtsH substrates to understand the function of FtsH during sporulation. I hypothesize that these protein substrates are supposed to be overproduced in an ftsH knockout and function during sporulation. As a result, among 28 proteins significantly increased in the absence of FtsH, the most abundant protein was identified as Spo0M with its predicted function as a sporulation control gene. The Spo0M level increased about 4.5-fold in the ftsH null mutant (Fig. 3.1).
The spo0M gene has been shown to control sporulation during the process from stage 0 to stage II (Han et al., 1998). An σH-like promoter has been detected in the upstream region of spo0M, and it has also been shown to be down-regulated by benzoate at pH 7.0 or by a low external pH (Kitko et al., 2009). A spo0M null mutant is viable,
Results
blocked at stage 0, and its sporulation frequency is reduced by 20- to 100-fold. If the spo0M gene is inserted into a high-copy number plasmid, the sporulation frequency is reduced, indicating that overproduction of the Spo0M protein results in a negative effect on sporulation (Han et al., 1998).
Since Spo0M is overproduced at the beginning of stage 0 in the absence of FtsH, we first asked whether FtsH regulates expression of Spo0M directly or indirectly.
Figure 3.1. Comparative proteomics of a wild type ftsH and null mutant strain. (A) Strains 1012 (ftsH+) and (B) WW01 (ftsH::erm) were grown in DSM to stage 0 at 37°C.
Then, intracellular proteins were separated by 2D electrophoresis. Proteins were separated by a pH gradient of 4 to 7 in the first dimension followed by the second dimension separation of SDS-PAGE. Gels were stained by Coomassie brilliant blue, and the protein spot of Spo0M is indicated.